Summary of "Spektrofotometri UV Vis Kuantitatif"

Purpose

Demonstrate a complete quantitative UV‑Vis spectrophotometry practical using salicylic acid: preparation of stock and calibration standards, sample preparation, instrument setup, measurement, data processing (standard curve), and cleanup.

Core lessons

Materials and preparation

Tools and reagents mentioned:

Detailed methodology — step‑by‑step

Stock solution (100 mL)

  1. Clean the analytical balance chamber with a brush before use.
  2. Weigh 100 mg (0.1 g) salicylic acid on weighing paper using the analytical balance.
  3. Clean the balance after weighing.
  4. Transfer the 100 mg into a 100 mL volumetric flask.
  5. Add ~25 mL ethanol and swirl to dissolve (homogenize).
  6. When dissolved, bring the volume up to the 100 mL mark with ethanol. Use a dropper when near the meniscus to reach the mark precisely.
  7. Homogenize the final stock solution (invert/mix).

Calibration standards (prepared in 25 mL flasks)

Sample preparation (to obtain sample B from original sample)

  1. Weigh sample: dissolve 100 mg salicylic acid sample in 10 mL ethanol in a beaker; stir to dissolve.
  2. Filter the mixture through filter paper into a container. (Attach filter paper to the funnel using water to hold it in place if needed.)
  3. Rinse the beaker with two 10 mL portions of ethanol and filter these rinses into the same flask (total rinses: 2 × 10 mL).
  4. Combine filtrate and rinses into a 50 mL volumetric flask and dilute to the mark with ethanol — this is solution A.
  5. Prepare solution B by taking 500 μL of solution A with a micropipette and diluting to 25 mL in a volumetric flask.
  6. Label sample replicates as B1, B2, B3.

Instrument setup and blanking (UV‑Vis)

  1. On the spectrophotometer software, change the method to photometric mode.
  2. Set the measurement wavelength to 297 nm (previously determined λmax).
  3. Use multi‑point calibration settings as required (enter 297 nm).
  4. Prepare the blank: ethanol is used as the blank solvent.
  5. Rinse the cuvette with blank (front and back), then fill the cuvette with blank and wipe clear surfaces with tissue.
  6. Insert the cuvette so the clear (optical) faces align with the instrument axis (clear faces the X axis; opaque faces the Y axis/toward operator as instructed).
  7. Run auto‑zero and baseline before measuring standards.

Measurement procedure

Standards

  1. Replace the blank with each standard, starting with the 200 μL standard.
    • Rinse the cuvette with the sample first (rinse until about 1/4 full using a circular motion), discard the rinse, then fill to ~3/4 for measurement.
  2. Read and record absorbance for each standard (200 → 700 μL series).
  3. After measuring all standards, generate the standard curve (absorbance vs concentration).
  4. Display the linear regression equation and R² (right‑click properties → show equation and R²).

Unknown sample (sample B)

  1. Rinse cuvette with sample B, then fill and place in instrument with correct orientation.
  2. Run “read unknown” in the software; the absorbance and calculated concentration (based on the calibration) will appear.
  3. Repeat for three replicates (B1, B2, B3) to assess reproducibility.

Data processing and saving/printing

Cleanup and shutdown

Practical tips emphasized: - Use a dropper for final volume adjustments to match the meniscus precisely. - Always homogenize after preparing or diluting solutions. - Rinse cuvettes with the solution to be measured before filling for the actual measurement to avoid contamination. - Use correct cuvette orientation for accurate readings. - Run auto zero and baseline before measurements to ensure correct baseline and zeroing. - Label replicate samples (B1–B3) and perform replicates for reliability. - Filter samples and rinse funnel/beaker to avoid loss and ensure complete transfer.

Speakers / sources

Category ?

Educational

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